The laccase (P-diphenol oxidase, EC 1.10.3.2) is a multi-copper-binding protein, belonging to coveline oxidoase, which may be derived from fungi (e.g. mushrooms), bacteria and plants, especially presented widely in white rot fungi of basidiomycetes. Laccase can be lived in the air, and is a environment-friendly ferment whose sole product is water after reaction. Due to recognizing environment protection gradually, recently laccase is hot point at home and abroad. Fungi laccases have four copper ion in general, so its electric levels of oxidation and deoxidization is higher than laccase from insects and plants. The laccase can oxidize several kinds of hydroxybenzene, arylamine, carboxylic acid, steroid hormone, biochrome, metallorganic compound and some non-phenolic substrates. So it can be applied widely in several modern industrial fields such as waste water treatment, paper pulp blanching, soil cleansing, dye decoloring, immunity detection, etc. Lentinus edodus (Lentinula edodes) is also named Araricus bretschneideri Kalich et Thum, belonging to Bssidiomycetes class, Agaricales order, Mushroom family, Leather pleat L. genus, whose formal name is Fragrant leather wimple bacteria. Lentinus edodus has longtime history of production and cultivation in our country, where is biggest country in globe of Lentinus edodus production. Now Lentinus edodus have been cultivated in more than 70% of provinces of our country. So Lentinus edodus has some characters such as low price, more producing areas, easily cultivation. As a kind of white rot fungi, Lentinus edodus has been proved that it can be used to produce laccase, which includes several kinds of isoenzyme and is collectively named by total laccase. And the activity of oxidation and deoxidization of the laccase is also much higher than that of the laccase from insects or plants. Comparing with extracting directly from Lentinus edodus sporophore, liquid fermentation technology with low cost and higher efficiency is presently the developing direction of industrial fermentation production. However, there is too few researches related to Lentinus edodus fermentation to produce laccase by now. The yield of Lentinus edodus laccase is still lower than that of other active components of Lentinus edodus by fermentation and extract. And the enzyme activity of laccase is restricted by many factors such as culture conditions and substrate components. It has been reported that some Aromatic compounds or heavy metal ion may be used to induce and increase the outputs, but these inducer are commonly toxic and difficult to degrade. After adding these inducer, the cost of treating fermentation liquid is increasing and the environment is also easily polluted. Using static liquid culturing or solid fermentation technologies may relieve the problem of environment pollution of laccase production, but the fermentation period is too long, which is not suitable for industrial fermentation production.
Chinese patent CN1463578A (application No. 02124094) discloses one kind of deep fermentation method of culturing liquid Lentinus edodes seed and its culture medium. The culture method includes the steps of: (1) performing mother seed culture in a mother seed culture medium; (2) transferring the mycelia overgrowing in the mother seed medium of the tubes to original sawdust culture medium for transitional culturing at temperature 22˜27±1° C. for 12˜20 days in dark until mycelium overgrowing; (3) transferring the fully crushed sawdust seed immediately to the liquid culture medium for amplification in the first shake flask; (4) transferring the liquid culture cultured for 5-7 days in the first shake flask to the second shake flask with fresh liquid culture medium for amplification; (5) and treating the liquid culture cultured in the second shake flask by fermentation to obtain liquid Lentinus edodus seed product. The yields using other laccase gene for heterogenous expression are lower than wild type. These factors limit the industrial production and application of laccase. So it is much needed to develop a new preparation method and production process of increasing production ratio of laccase and decreasing production cost to satisfy modern industry.
Recently a new type of protein named expansin is found in plant cell walls. Studies have shown that expansin plays an very important role in several aspects such as plant morphogenesis (Fleming et al., 1999), fruit ripening (Cosgrove, 2000), root hair formation (Cho and Cosgrove, 2002), cell expanding (Cosgrove, 1998), penetration regulation (Wu and Cosgrove, 2000) and pollen tube growth of graminaceous plants (Cosgrove, 1997), etc. Expansin has been found in Arabidopsis, tomato, strawberry, cotton, rice, corn and other plants, and is considered existing in various dicotyledonous and monocotyledonous plant cell walls. Experiments have shown that as a plant cell wall protein, expansin has the function of recovering the thermal inactivated cell wall to extend in vitro. Therefore, it is supposed that expansin can regulate physiological activities such as acid-dependent cell wall extension and stress relaxation by breaking the hydrogen bonds between the cell wall polymers, and may play regulation functions such as physiological regulation and cell wall extension process in the period of plant growth. However, the mechanism of expansin is not yet a clear conclusion. Currently it is not reported at home and abroad that the expansin is applied in liquid fermentation of Lentinus edodes to produce secondary metabolites such as laccase.